Tobacco products with increased nicotine

ABSTRACT

DNA encoding a plant quinolate phosphoribosyl transferase (QPRTase) enzyme, and constructs comprising such DNA are provided. Methods of altering quinolate phosphoribosyl transferase expression are provided.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation application of, and claims priorityto, U.S. Application Ser. No. 10/356,076, filed Jan. 3, 2003, whichstatus is pending, which is a continuation application of U.S.Application Ser. No. 09/021,286, filed Feb. 10, 1998 and issued as U.S.Pat. No. 6,586,661 on Jul. 1, 2003 and which claims the benefit of U.S.Provisional Application No. 60/049,471, filed Jun. 12, 1997. The entirecontents of each of these applications is incorporated by referenceherein.

FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under National ScienceFoundation Grant No. MCB-9206506. The Government has certain rights tothis invention.

FIELD OF THE INVENTION

This invention relates to plant quinolate phosphoribosyl transferase(QPRTase) and to DNA encoding this enzyme. In particular, this inventionrelates to the use of DNA encoding quinolate phosphoribosyl transferaseto produce transgenic plants having genetically altered nicotine levels,and the plants so produced.

BACKGROUND OF THE INVENTION

The production of tobacco with decreased levels of nicotine is ofinterest, given concerns regarding the addictive nature of nicotine.Additionally, tobacco plants with extremely low levels of nicotineproduction, or no nicotine production, are attractive as recipients fortransgenes expressing commercially valuable products such aspharmaceuticals, cosmetic components, or food additives. Variousprocesses have been designed for the removal of nicotine from tobacco.However, most of these processes remove other ingredients from tobaccoin addition to nicotine, thereby adversely affecting the tobacco.Classical crop breeding techniques have produced tobacco plants withlower levels of nicotine (approximately 8%) than that found in wild-typetobacco plants. Tobacco plants and tobacco having even furtherreductions in nicotine content are desirable.

One approach for reducing the level of a biological product is to reducethe amount of a required enzyme in the biosynthetic pathway leading tothat product. Where the affected enzyme naturally occurs in arate-limiting amount (relative to the other enzymes required in thepathway), any reduction in that enzyme's abundance will decrease theproduction of the end product. If the amount of the enzyme is notnormally rate limiting, its presence in a cell must be reduced torate-limiting levels in order to diminish the pathway's output.Conversely, if the naturally-occurring amount of enzyme is ratelimiting, then any increase in the enzyme's activity will result in anincrease in the biosynthetic pathway's end product.

Nicotine is formed primarily in the roots of the tobacco plant and issubsequently transported to the leaves, where it is stored (Tso,Physiology and Biochemistry of Tobacco Plants, pp. 233-34, Dowden,Hutchinson & Ross, Stroudsburg, Pa. (1972)). An obligatory step innicotine biosynthesis is the formation of nicotinic acid from quinolinicacid, which step is catalyzed by the enzyme quinoline phosphoribosyltransferase (“QPRTase”). QPRTase appears to be a rate-limiting enzyme inthe pathway supplying nicotinic acid for nicotine synthesis in tobacco.See, e.g., Feth et al., “Regulation in Tobacco Callus of EnzymeActivities of the Nicotine Pathway”, Planta, 168, pp. 402-07 (1986);Wagner et al., “The Regulation of Enzyme Activities of the NicotinePathway in Tobacco”, Physiol. Plant., 68, pp. 667-72 (1986). Themodification of nicotine levels in tobacco plants by antisenseregulation of putrescence methyl transferase (PMTase) expression isproposed in U.S. Pat. Nos. 5,369,023 and 5,260,205 to Nakatani andMalik. PCT application WO 94/28142 to Wahad and Malik 30 describes DNAencoding PMT and the use of sense and antisense PMT constructs.

SUMMARY OF THE INVENTION

A first aspect of the present invention is an isolated DNA moleculecomprising SEQ ID NO:1; DNA sequences which encode an enzyme having SEQID NO:2; DNA sequences which hybridize to such DNA and which encode aquinolate phosphoribosyl transferase enzyme; and DNA sequences whichdiffer from the above DNA due to the degeneracy of the genetic code. Apeptide encoded by such DNA is a further aspect of the invention.

A further aspect of the present invention is a DNA construct comprisinga promoter operable in a plant cell and a DNA segment encoding aquinolate phosphoribosyl transferase enzyme positioned downstream fromthe promoter and operatively associated therewith. The DNA encoding theenzyme may be in the antisense or sense direction.

A further aspect of the present invention is a method of makingtransgenic plant cell having reduced quinolate phosphoribosyltransferase (QPRTase) expression, by providing a plant cell of a typeknown to express quinolate phosphoribosyl transferase; transforming theplant cell with an exogenous DNA construct comprising a promoter and DNAcomprising a portion of a sequence encoding quinolate phosphoribosyltransferase mRNA.

A further aspect of the present invention is a transgenic plant of thespecies Nicotiana having reduced quinolate phosphoribosyl transferase(QPRTase) expression relative to a non-transformed control plant. Thecells of such plants comprise a DNA construct which includes a segmentof a DNA sequence that encodes a plant quinolate phosphoribosyltransferase mRNA.

A further aspect of the present invention is a method for reducingexpression of a quinolate phosphoribosyl transferase gene in a plantcell by growing a plant cell transformed to contain exogenous DNA, wherea transcribed strand of the exogenous DNA is complementary to quinolatephosphoribosyl transferase mRNA endogenous to the cell. Transcription ofthe complementary strand reduces expression of the endogenous quinolatephosphoribosyl gene.

A further aspect of the present invention is a method of producing atobacco plant having decreased levels of nicotine in leaves of thetobacco plant by growing a tobacco plant with cells that comprise anexogenous DNA sequence, where a transcribed strand of the exogenous DNAsequence is complementary to endogenous quinolate phosphoribosyltransferase messenger RNA in the cells.

A further aspect of the present invention is a method of making atransgenic plant cell having increased quinolate phosphoribosyltransferase (QPRTase) expression, by transforming a plant cell known toexpress quinolate phosphoribosyl transferase with an exogenous DNAconstruct which comprises a DNA sequence encoding quinolatephosphoribosyl transferase.

A further aspect of the present invention is a transgenic Nicotianaplant having increased quinolate phosphoribosyl transferase (QPRTase)expression, where cells of the transgenic plant comprise an exogenousDNA sequence encoding a plant quinolate phosphoribosyl transferase.

A further aspect of the present invention is a method for increasingexpression of a quinolate phosphoribosyl transferase gene in a plantcell, by growing a plant cell transformed to contain exogenous DNAencoding quinolate phosphoribosyl transferase.

A further aspect of the present invention is a method of producing atobacco plant having increased levels of nicotine in the leaves, bygrowing a tobacco plant having cells that contain an exogenous DNAsequence that encodes quinolate phosphoribosyl transferase functional inthe cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the biosynthetic pathway leading to nicotine. Enzymeactivities known to be regulated by Nic1 and Nic2 are QPRTase (quinolatephosphoribosyl transferase) and PMTase (putrescence methyltransferase).

FIG. 2A provides the nucleic acid sequence of NtQPT1 cDNA (SEQ ID NO:1),with the coding sequence (SEQ ID NO:3) shown in capital letters.

FIG. 2B provides the deduced amino acid sequence (SEQ ID NO:2) of thetobacco QPRTase encoded by NtQPT1 cDNA.

FIG. 3 aligns the deduced NtQPT1 amino acid sequence and relatedsequences of Rhodospirillum rubrum, Mycobacterium lepre, Salmonellatyphimurium, Escherichia coli, human, and Saccharomyces cerevisiae.

FIG. 4 shows the results of complementation of an Escherichia colimutant lacking quinolate phosphoribosyl transferase (TH265) with NTQPT1cDNA. Cells were transformed with an expression vector carrying NtQPT1;growth of transformed TH265 cells expressing NtQPT1 on minimal mediumlacking nicotinic acid demonstrated that NtQPT1 encodes QPRTase.

FIG. 5 compares nicotine levels and the relative steady-state NtQTP1mRNA levels in Nic1 and Nic2 tobacco mutants: wild-type Burley 21(Nic1/Nic1 Nic2/Nic2); Nic1⁻ Burley 21 (nic1/nic] Nic2/Nic2); Nic2⁻Burley 21 (Nic1/Nic1 nic2/nic2); and Nic1⁻ Nic2⁻ Burley 21 (nic1/nic1nic2/nic2). Solid bars indicate mRNA transcript levels; hatched barsindicate nicotine levels.

FIG. 6 charts the relative levels of NtQPT1 mRNA over time in toppedtobacco plants compared to non-topped control plants. Solid barsindicate mRNA transcript levels; hatched bars indicate nicotine levels.

DETAILED DESCRIPTION OF THE INVENTION

Nicotine is produced in tobacco plants by the condensation of nicotinicacid and 4-methylaminobutanal. The biosynthetic pathway resulting innicotine production is illustrated in FIG. 1. Two regulatory loci (Nic1and Nic2) act as co-dominant regulators of nicotine production. Enzymeanalyses of roots of single and double Nic mutants show that theactivities of two enzymes, quinolate phosphoribosyl transferase(QPRTase) and putrescence methyl transferase (PMTase), are directlyproportional to levels of nicotine biosynthesis. A comparison of enzymeactivity in tobacco tissues (root and callus) with different capacitiesfor nicotine synthesis shows that QPRTase activity is strictlycorrelated with nicotine content (Wagner and Wagner, Planta 165:532(1985)). Saunders and Bush (Plant Physiol 64:236 (1979) showed that thelevel of QPRTase in the roots of low nicotine mutants is proportional tothe levels of nicotine in the leaves.

The present invention encompasses a novel cDNA sequence (SEQ ID NO:1)encoding a plant quinolate phosphoribosyl transferase (QPRTase) of SEQID NO:2. As QPRTase activity is strictly correlated with nicotinecontent, construction of transgenic tobacco plants in which QPRTaselevels are lowered in the plant roots (compared to levels in wild-typeplants) result in plants having reduced levels of nicotine in theleaves. The present invention provides methods and nucleic acidconstructs for producing such transgenic plants, as well as suchtransgenic plants. Such methods include the expression of antisenseNtQPT1 RNA, which lowers the amount of QPRTase in tobacco roots.Nicotine has additionally been found in non-tobacco species and familiesof plants, though the amount present is usually much lower than in N.tabacum.

The present invention also provides sense and antisense recombinant DNAmolecules encoding QPRTase or QPRTase antisense RNA molecules, andvectors comprising those recombinant DNA molecules, as well astransgenic plant cells and plants transformed with those DNA moleculesand vectors. Transgenic tobacco cells and plants of this invention arecharacterized by lower or higher nicotine content than untransformedcontrol tobacco cells and plants.

Tobacco plants with extremely low levels of nicotine production, or nonicotine production, are attractive as recipients for transgenesexpressing commercially valuable products such as pharmaceuticals,cosmetic components, or food additives. Tobacco is attractive as arecipient plant for a transgene encoding a desirable product, as tobaccois easily genetically engineered and produces a very large biomass peracre; tobacco plants with reduced resources devoted to nicotineproduction accordingly will have more resources available for productionof transgene products. Methods of transforming tobacco with transgenesproducing desired products are known in the art; any suitable techniquemay be utilized with the low nicotine tobacco plants of the presentinvention.

Tobacco plants according to the present invention with reduced QPRTaseexpression and reduced nicotine levels will be desirable in theproduction of tobacco products having reduced nicotine content. Tobaccoplants according to the present invention will be suitable for use inany traditional tobacco product, including but not limited to pipe,cigar and cigarette tobacco, and chewing tobacco, and may be in any formincluding leaf tobacco, shredded tobacco, or cut tobacco.

The constructs of the present invention may also be useful in providingtransgenic plants having increased QPRTase expression and increasednicotine content in the plant. Such constructs, methods using theseconstructs and the plants so produced may be desirable in the productionof tobacco products having altered nicotine content, or in theproduction of plants having nicotine content increased for itsinsecticidal effects.

The present inventors have discovered that the TobRD2 gene (see Conklinget al., Plant Phys. 93, 1203 (1990)) encodes a Nicotiana 20 tabacumQPRTase, and provide herein the cDNA sequence of NTQPT1 (formerly termedTobRD2) and the amino acid sequence of the encoded enzyme. Comparisonsof the NtQPT1 amino acid sequence with the GenBank database reveallimited sequence similarity to bacterial proteins that encode quinolatephosphoribosyl transferase (QPRTase) (FIG. 3).

Quinolate phosphoribosyl transferase is required for de novo nicotineadenine dinucleotide (NAD) biosynthesis in both prokaryotes andeukaryotes. In tobacco, high levels of QPRTase are detected in roots,but not in leaves. To determine that NtQPT1 encoded QPRTase, the presentinventors utilized Escherichia coli bacterial strain (TH265), a mutantlacking in quinolate phosphoribosyl transferase (nadC⁻). This mutantcannot grow on minimal medium lacking nicotinic acid. However,expression of the NtQPT1 protein in this bacterial strain conferred theNadC⁺ phenotype (FIG. 4), confirming that NIQPT1 encodes QPRTase.

The present inventors examined the effects of Nic1 and Nic2 mutants intobacco, and the effects of topping tobacco plants, on NtQPT1steady-state mRNA levels and nicotine levels. (Removal of apicaldominance by topping at onset of flowering is well known to result inincreased levels of nicotine biosynthesis and transport in tobacco, andis a standard practice in tobacco production.) If NtQPT1 is in factinvolved in nicotine biosynthesis, it would be expected that (1) NtQPT1mRNA levels would be lower in Nic1/Nic2 double mutants and (2) NtQPT1mRNA levels would increase after topping. NtQPT1 mRNA levels inNic1/Nic2 double mutants were found to be approximately 25% that ofwild-type (FIG. 5). Further, within six hours of topping, the NTQPT1mRNA levels in tobacco plants increased about eight-fold. Therefore,NtQPT1 was determined to be a key regulatory gene in the nicotinebiosynthetic pathway.

Transgenic Plant Cells and Plants

Regulation of gene expression in plant cell genomes can be achieved byintegration of heterologous DNA under the transcriptional control of apromoter which is functional in the host, and in which the transcribedstrand of heterologous DNA is complementary to the strand of DNA that istranscribed from the endogenous gene to be regulated. The introducedDNA, referred to as antisense DNA, provides an RNA sequence which iscomplementary to naturally produced (endogenous) mRNAs and whichinhibits expression of the endogenous mRNA. The mechanism of such geneexpression regulation by antisense is not completely understood. Whilenot wishing to be held to any single theory, it is noted that one theoryof antisense regulation proposes that transcription of antisense DNAproduces RNA molecules which bind to and prevent or inhibittranscription of endogenous mRNA molecules.

In the methods of the present invention, the antisense product may becomplementary to coding or non-coding (or both) portions of naturallyoccurring target RNA. The antisense construction may be introduced intothe plant cells in any suitable manner, and may be integrated into theplant genome for inducible or constitutive transcription of theantisense sequence. See, e.g., U.S. Pat. Nos. 5,453,566 and 5,107,065 toShewmaker et al. (incorporated by reference herein in their entirety).As used herein, exogenous or heterologous DNA (or RNA) refers to DNA (orRNA) which has been introduced into a cell (or the cell's ancestor)through the efforts of humans. Such heterologous DNA may be a copy of asequence which is naturally found in the cell being transformed, orfragments thereof.

To produce a tobacco plant having decreased QPRTase levels, and thuslower nicotine content, than an untransformed control tobacco plant, atobacco cell may be transformed with an exogenous QPRT antisensetranscriptional unit comprising a partial QPRT cDNA sequence, afull-length QPRT cDNA sequence, a partial QPRT chromosomal sequence, ora full-length QPRT chromosomal sequence, in the antisense orientationwith appropriate operably linked regulatory sequences. Appropriateregulatory sequences include a transcription initiation sequence(“promoter”) operable in the plant being transformed, and apolyadenylation/transcription termination sequence. Standard techniques,such as restriction mapping,. Southern blot hybridization, andnucleotide sequence analysis, are then employed to identify clonesbearing QPRTase sequences in the antisense orientation, operably linkedto the regulatory sequences. Tobacco plants are then regenerated fromsuccessfully transformed cells. It is most preferred that the antisensesequence utilized be complementary to the endogenous sequence, however,minor variations in the exogenous and endogenous sequences may betolerated. It is preferred that the antisense DNA sequence be ofsufficient sequence similarity that it is capable of binding to theendogenous sequence in the cell to be regulated, under stringentconditions as described below.

Antisense technology has been employed in several laboratories to createtransgenic plants characterized by lower than normal amounts of specificenzymes. For example, plants with lowered levels of chalcone synthase,an enzyme of a flower pigment biosynthetic pathway, have been producedby inserting a chalcone synthase antisense gene into the genome oftobacco and petunia. These transgenic tobacco and petunia plants produceflowers with lighter than normal coloration (Van der Krol et al., “AnAnti-Sense Chalcone Synthase Gene in Transgenic Plants Inhibits FlowerPigmentation”, Nature, 333, pp. 866-69 (1988)). Antisense RNA technologyhas also been successfully employed to inhibit production of the enzymepolygalacturonase in tomatoes (Smith et al., “Antisense RNA Inhibitionof Polygalacturonase Gene Expression in Transgenic Tomatoes”, Nature,334, pp. 724-26 (1988); Sheehy et al., “Reduction of PolygalacturonaseActivity in Tomato Fruit by Antisense RNA”, Proc. Nat. Acad Sci. USA,85, pp. 8805-09 (1988)), and the small subunit of the enzyme ribulosebisphosphate carboxylase in tobacco (Rodermel et al., “Nuclear-OrganelleInteractions: Nuclear Antisense Gene Inhibits Ribulose BisphosphateCarboxylase Enzyme Levels in Transformed Tobacco Plants”, Cell, 55, pp.673-81 (1988)). Alternatively, transgenic plants characterized bygreater than normal amounts of a given enzyme may be created bytransforming the plants with the gene for that enzyme in the sense(i.e., normal) orientation. Levels of nicotine in the transgenic tobaccoplants of the present invention can be detected by standard nicotineassays. Transformed plants in which the level of QPRTase is reducedcompared to untransformed control plants will accordingly have a reducednicotine level compared to the control; transformed plants in which thelevel of QPRTase is increased compared to untransformed control plantswill accordingly have an increased nicotine level compared to thecontrol.

The heterologous sequence utilized in the antisense methods of thepresent invention may be selected so as to produce an RNA productcomplementary to the entire QPRTase mRNA sequence, or to a portionthereof. The sequence may be complementary to any contiguous sequence ofthe natural messenger RNA, that is, it may be complementary to theendogenous mRNA sequence proximal to the 5′-terminus or capping site,downstream from the capping site, between the capping site and theinitiation codon and may cover all or only a portion of the non-codingregion, may bridge the non-coding and coding region, be complementary toall or part of the coding region, complementary to the 3′-terminus ofthe coding region, or complementary to the 3′-untranslated region of themRNA. Suitable antisense sequences may be from at least about 13 toabout 15 nucleotides, at least about 16 to about 21 nucleotides, atleast about 20 nucleotides, at least about 30 nucleotides, at leastabout 50 nucleotides, at least about 75 nucleotides, at least about 100nucleotides, at least about 125 nucleotides, at least about 150nucleotides, at least about 200 nucleotides, or more. In addition, thesequences may be extended or shortened on the 3′ or 5′ ends thereof.

The particular anti-sense sequence and the length of the anti-sensesequence will vary depending upon the degree of inhibition desired, thestability of the anti-sense sequence, and the like. One of skill in theart will be guided in the selection of appropriate QPRTase antisensesequences using techniques available in the art and the informationprovided herein. With reference to FIG. 2A and SEQ ID NO: 1 herein, anoligonucleotide of the invention may be a continuous fragment of theQPRTase cDNA sequence in antisense orientation, of any length that issufficient to achieve the desired effects when transformed into arecipient plant cell.

The present invention may also be used in methods of senseco-suppression of nicotine production. Sense DNAs employed in carryingout the present invention are of a length sufficient to, when expressedin a plant cell, suppress the native expression of the plant QPRTaseprotein as described herein in that plant cell. Such sense DNAs may beessentially an entire genomic or complementary DNA encoding the QPRTaseenzyme, or a fragment thereof with such fragments typically being atleast 15 nucleotides in length. Methods of ascertaining the length ofsense DNA that results in suppression of the expression of a native genein a cell are available to those skilled in the art.

In an alternate embodiment of the present invention, Nicotiana 30 plantcells are transformed with a DNA construct containing a DNA segmentencoding an enzymatic RNA molecule (i.e., a “ribozyme”), which enzymaticRNA molecule is directed against (i.e., cleaves) the mRNA transcript ofDNA encoding plant QPRTase as described herein. Ribozymes containsubstrate binding domains that bind to accessible regions of the targetmRNA, and domains that catalyze the cleavage of RNA, preventingtranslation and protein production. The binding domains may compriseantisense sequences complementary to the target mRNA sequence; thecatalytic motif may be a hammerhead motif or other motifs, such as thehairpin motif. Ribozyme cleavage sites within an RNA target mayinitially be identified by scanning the target molecule for ribozymecleavage sites (e.g., GUA, GUU or GUC sequences). Once identified, shortRNA sequences of 15, 20, 30 or more ribonucleotides corresponding to theregion of the target gene containing the cleavage site may be evaluatedfor predicted structural features. The suitability of candidate targetsmay also be evaluated by testing their accessibility to hybridizationwith complimentary oligonucleotides, using ribonuclease protectionassays as are known in the art. DNA encoding enzymatic RNA molecules maybe produced in accordance with known techniques. See, e.g., T. Cech etal., U.S. Pat. No. 4,987,071; Keene et al., U.S. Pat. No. 5,559,021;Donson et al., U.S. Pat. No. 5,589,367; Torrence et al., U.S. Pat. No.5,583,032; Joyce, U.S. Pat. No. 5,580,967; Gold et al. U.S. Pat. No.5,595,877; Wagner et al., U.S. Pat. No. 5,591,601; and U.S. Pat. No.5,622,854 (the disclosures of which are to be incorporated herein byreference in their entirety). Production of such an enzymatic RNAmolecule in a plant cell and disruption of QPRTase protein productionreduces QPRTase activity in plant cells in essentially the same manneras production of an antisense RNA molecule: that is, by disruptingtranslation of mRNA in the cell which produces the enzyme. The term‘ribozyme’ is used herein to describe an RNA-containing nucleic acidthat functions as an enzyme (such as an endoribonuclease), and may beused interchangeably with ‘enzymatic RNA molecule’. The presentinvention further includes DNA encoding the ribozymes, DNA encodingribozymes which has been inserted into an expression vector, host cellscontaining such vectors, and methods of decreasing QPRTase production inplants using ribozymes.

Nucleic acid sequences employed in carrying out the present inventioninclude those with sequence similarity to SEQ ID NO:1, and encoding aprotein having quinolate phosphoribosyl transferase activity. Thisdefinition is intended to encompass natural allelic variations inQPRTase proteins. Thus, DNA sequences that hybridize to DNA of SEQ IDNO:1 and code for expression of QPRTase, particularly plant QPRTaseenzymes, may also be employed in carrying out the present invention.

Multiple forms of tobacco QPRT enzyme may exist. Multiple forms of anenzyme may be due to post-translational modification of a single geneproduct, or to multiple forms of the NTQPT1 gene.

Conditions which permit other DNA sequences which code for expression ofa protein having QPRTase activity to hybridize to DNA of SEQ ID NO:1 orto other DNA sequences encoding the protein given as SEQ ID NO:2 can bedetermined in a routine manner. For example, hybridization of suchsequences may be carried out under conditions of reduced stringency oreven stringent conditions (e.g., conditions represented by a washstringency of 0.3 M NaCl, 0.03 M sodium citrate, 0.1% SDS at 60° C. oreven 70° C. to DNA encoding the protein given as SEQ ID NO:2 herein in astandard in situ hybridization assay. See J. Sambrook et al., MolecularCloning, A Laboratory Manual (2d Ed. 1989)(Cold Spring HarborLaboratory)). In general, such sequences will be at least 65% similar,75% similar, 80% similar, 85% similar, 90% similar, or even 95% similar,or more, with the sequence given herein as SEQ ID NO:1, or DNA sequencesencoding proteins of SEQ ID NO:2. (Determinations of sequence similarityare made with the two sequences aligned for maximum matching; gaps ineither of the two sequences being matched are allowed in maximizingmatching. Gap lengths of 10 or less are preferred, gap lengths of 5 orless are more preferred, and gap lengths of 2 or less still morepreferred.)

Differential hybridization procedures are available which allow for theisolation of cDNA clones whose mRNA levels are as low as about 0.05% ofpoly(A⁺)RNA. See M. Conkling et al., Plant Physiol. 93, 1203-1211(1990). In brief, cDNA libraries are screened using single-stranded cDNAprobes of reverse transcribed mRNA from plant tissue (e.g., roots and/orleaves). For differential screening, a nitrocellulose or nylon membraneis soaked in 5×SSC, placed in a 96 well suction manifold, 150 μL ofstationary overnight culture transferred from a master plate to eachwell, and vacuum applied until all liquid has passed through the filter.150 μL of denaturing solution (0.5M NaOH, 1.5 M NaCl) is placed in eachwell using a multiple pipetter and allowed to sit about 3 minutes.Suction is applied as above and the filter removed and neutralized in0.5 M Tris-HCl (pH 8.0), 1.5 M NaCl. It is then baked 2 hours in vacuoand incubated with the relevant probes. By using nylon membrane filtersand keeping master plates stored at −70° C. in 7% DMSO, filters may bescreened multiple times with multiple probes and appropriate clonesrecovered after several years of storage.

As used herein, the term ‘gene’ refers to a DNA sequence thatincorporates (1) upstream (5′) regulatory signals including thepromoter, (2) a coding region specifying the product, protein or RNA ofthe gene, (3) downstream (3′) regions including transcriptiontermination and polyadenylation signals and (4) associated sequencesrequired for efficient and specific expression.

The DNA sequence of the present invention may consist essentially of thesequence provided herein (SEQ ID NO:1), or equivalent nucleotidesequences representing alleles or polymorphic variants of these genes,or coding regions thereof.

Use of the phrase “substantial sequence similarity” in the presentspecification and claims means that DNA, RNA or amino acid sequenceswhich have slight and non-consequential sequence variations from theactual sequences disclosed and claimed herein are considered to beequivalent to the sequences of the present invention. In this regard,“slight and non-consequential sequence variations” mean that “similar”sequences (i.e., the sequences that have substantial sequence similaritywith the DNA, RNA, or proteins disclosed and claimed herein) will befunctionally equivalent to the sequences disclosed and claimed in thepresent invention. Functionally equivalent sequences will function insubstantially the same manner to produce substantially the samecompositions as the nucleic acid and amino acid compositions disclosedand claimed herein.

DNA sequences provided herein can be transformed into a variety of hostcells. A variety of suitable host cells, having desirable growth andhandling properties, are readily available in the art.

Use of the phrase “isolated” or “substantially pure” in the presentspecification and claims as a modifier of DNA, RNA, polypeptides orproteins means that the DNA, RNA, polypeptides or proteins so designatedhave been separated from their in vivo cellular environments through theefforts of human beings. As used herein, a “native DNA sequence” or“natural DNA sequence” means a DNA sequence which can be isolated fromnon-transgenic cells or tissue. Native DNA sequences are those whichhave not been artificially altered, such as by site-directedmutagenesis. Once native DNA sequences are identified, DNA moleculeshaving native DNA sequences may be chemically synthesized or producedusing recombinant DNA procedures as are known in the art. As usedherein, a native plant DNA sequence is that which can be isolated fromnon-transgenic plant cells or tissue. As used herein, a native tobaccoDNA sequence is that which can be isolated from non-transgenic tobaccocells or tissue DNA constructs, or “transcription cassettes,” of thepresent invention include, 5, to 3′ in the direction of transcription, apromoter as discussed herein, a DNA sequence as discussed hereinoperatively associated with the promoter, and, optionally, a terminationsequence including stop signal for RNA polymerase and a polyadenylationsignal for polyadenylase. All of these regulatory regions should becapable of operating in the cells of the tissue to be transformed. Anysuitable termination signal may be employed in carrying out the presentinvention, examples thereof including, but not limited to, the nopalinesynthase (nos) terminator, the octapine synthase (ocs) terminator, theCaMV terminator, or native termination signals derived from the samegene as the transcriptional initiation region or derived from adifferent gene. See, e.g., Rezian et al. (1988) supra, and Rodermel etal. (1988), supra.

The term “operatively associated,” as used herein, refers to DNAsequences on a single DNA molecule which are associated so that thefunction of one is affected by the other. Thus, a promoter isoperatively associated with a DNA when it is capable of affecting thetranscription of that DNA (i.e., the DNA is under the transcriptionalcontrol of the promoter). The promoter is said to be “upstream” from theDNA, which is in turn said to be “downstream” from the promoter.

The transcription cassette may be provided in a DNA construct which alsohas at least one replication system. For convenience, it is common tohave a replication system functional in Escherichia coli, such as ColE1,pSCl01, pACYC184, or the like. In this manner, at each stage after eachmanipulation, the resulting construct may be cloned, sequenced, and thecorrectness of the manipulation determined. In addition, or in place ofthe E. coli replication system, a broad host range replication systemmay be employed, such as the replication systems of the P-1incompatibility plasmids, e.g., pRK290. In addition to the replicationsystem, there will frequently be at least one marker present, which maybe useful in one or more hosts, or different markers for individualhosts. That is, one marker may be employed for selection in aprokaryotic host, while another marker may be employed for selection ina eukaryotic host, particularly the plant host. The markers may beprotection against a biocide, such as antibiotics, toxins, heavy metals,or the like; may provide complementation, by imparting prototrophy to anauxotrophic host; or may provide a visible phenotype through theproduction of a novel compound in the plant.

The various fragments comprising the various constructs, transcriptioncassettes, markers, and the like may be introduced consecutively byrestriction enzyme cleavage of an appropriate replication system, andinsertion of the particular construct or fragment into the availablesite. After ligation and cloning the DNA construct may be isolated forfurther manipulation. All of these techniques are amply exemplified inthe literature as exemplified by J. Sambrook et al., Molecular Cloning,A Laboratory Manual (2d Ed. 1989)(Cold Spring Harbor Laboratory).

Vectors which may be used to transform plant tissue with nucleic acidconstructs of the present invention include both Agrobacterium vectorsand ballistic vectors, as well as vectors suitable for DNA-mediatedtransformation.

The term ‘promoter’ refers to a region of a DNA sequence thatincorporates the necessary signals for the efficient expression of acoding sequence. This may include sequences to which an RNA polymerasebinds but is not limited to such sequences and may include regions towhich other regulatory proteins bind together with regions involved inthe control of protein translation and may include coding sequences.

Promoters employed in carrying out the present invention may beconstitutively active promoters. Numerous constitutively activepromoters which are operable in plants are available. A preferredexample is the Cauliflower Mosaic Virus (CaMV) 35S promoter which isexpressed constitutively in most plant tissues. In the alternative, thepromoter may be a root-specific promoter or root cortex specificpromoter, as explained in greater detail below.

Antisense sequences have been expressed in transgenic tobacco plantsutilizing the Cauliflower Mosaic Virus (CaMV) 35S promoter. See, e.g.,Cornelissen et al., “Both RNA Level and Translation Efficiency areReduced by Anti-Sense RNA in Transgenic Tobacco”, Nucleic Acids Res. 17,pp. 833-43 (1989); Rezaian et al., “Anti-Sense RNAs of Cucumber MosaicVirus in Transgenic Plants Assessed for Control of the Virus”, PlantMolecular Biology 11, pp. 463-71 (1988); Rodermel et al.,“Nuclear-Organelle Interactions: Nuclear Antisense Gene InhibitsRibulose Bisphosphate Carboxylase Enzyme Levels in Transformed TobaccoPlants”, Cell 55, pp. 673-81 (1988); Smith et al., “Antisense RNAInhibition of Polygalacturonase Gene Expression in Transgenic Tomatoes”,Nature 334, pp. 724-26 (1988); Van der Krol et al., “An Anti-SenseChalcone Synthase Gene in Transgenic Plants Inhibits FlowerPigmentation”, Nature 333, pp. 866-69 (1988).

Use of the CaMV 35S promoter for expression of QPRTase in thetransformed tobacco cells and plants of this invention is preferred. Useof the CaMV promoter for expression of other recombinant genes intobacco roots has been well described (Lam et al., “Site-SpecificMutations Alter In Vitro Factor Binding and Change Promoter ExpressionPattern in Transgenic Plants”, Proc. Nat. Acad. Sci. USA 86, pp. 7890-94(1989); Pulse et al. “Dissection of 5′ Upstream Sequences for SelectiveExpression of the Nicotiana plumbaginifolia rbcS-8B Gene”, Mol. Gen.Genet. 214, pp. 16-23(1988)).

Other promoters which are active only in root tissues (root specificpromoters) are also particularly suited to the methods of the presentinvention. See, e.g., U.S. Pat. No. 5,459,252 to Conkling et al.;Yamamoto et al., The Plant Cell, 3:371 (1991). The TobRD2 root-cortexspecific promoter may also be utilized. See, e.g., U.S. patentapplication Ser. No. 08/508,786, now allowed, to Conkling et al.; PCT WO9705261. All patents cited herein are intended to be incorporated hereinby reference in their entirety.

The QPRTase recombinant DNA molecules and vectors used to produce thetransformed tobacco cells and plants of this invention may furthercomprise a dominant selectable marker gene. Suitable dominant selectablemarkers for use in tobacco include, inter alia, antibiotic resistancegenes encoding neomycin phosphotransferase (NPTII), hygromycinphosphotransferase (HPT), and chloramphenicol acetyltransferase (CAT).Another well-known dominant selectable marker suitable for use intobacco is a mutant dihydrofolate reductase gene that encodesmethotrexate-resistant dihydrofolate reductase. DNA vectors containingsuitable antibiotic resistance genes, and the corresponding antibiotics,are commercially available.

Transformed tobacco cells are selected out of the surrounding populationof non-transformed cells by placing the mixed population of cells into aculture medium containing an appropriate concentration of the antibiotic(or other compound normally toxic to tobacco cells) against which thechosen dominant selectable marker gene product confers resistance. Thus,only those tobacco cells that have been transformed will survive andmultiply.

Methods of making recombinant plants of the present invention, ingeneral, involve first providing a plant cell capable of regeneration(the plant cell typically residing in a tissue capable of regeneration).The plant cell is then transformed with a DNA construct comprising atranscription cassette of the present invention (as described herein)and a recombinant plant is regenerated from the transformed plant cell.As explained below, the transforming step is carried out by techniquesas are known in the art, including but not limited to bombarding theplant cell with microparticles carrying the transcription cassette,infecting the cell with an Agrobacterium tumefaciens containing a Tiplasmid carrying the transcription cassette, or any other techniquesuitable for the production of a transgenic plant.

Numerous Agrobacterium vector systems useful in carrying out the presentinvention are known. For example, U.S. Pat. No. 4,459,355 discloses amethod for transforming susceptible plants, including dicots, with anAgrobacterium strain containing the Ti plasmid. The transformation ofwoody plants with an Agrobacterium vector is disclosed in U.S. Pat. No.4,795,855. Further, U.S. Pat. No. 4,940,838 to Schilperoort et al.discloses a binary Agrobacterium vector (i.e., one in which theAgrobacterium contains one plasmid having the vir region of a Ti plasmidbut no T region, and a second plasmid having a T region but no virregion) useful in carrying out the present invention.

Microparticles carrying a DNA construct of the present invention, whichmicroparticle is suitable for the ballistic transformation of a plantcell, are also useful for making transformed plants of the presentinvention. The microparticle is propelled into a plant cell to produce atransformed plant cell, and a plant is regenerated from the transformedplant cell. Any suitable ballistic cell transformation methodology andapparatus can be used in practicing the present invention. Exemplaryapparatus and procedures are disclosed in Sanford and Wolf, U.S. Pat.No. 4,945,050, and in Christou et al., U.S. Pat. No. 5,015,580. Whenusing ballistic transformation procedures, the transcription cassettemay be incorporated into a plasmid capable of replicating in orintegrating into the cell to be transformed. Examples of microparticlessuitable for use in such systems include 1 to 5 μm gold spheres. The DNAconstruct may be deposited on the microparticle by any suitabletechnique, such as by precipitation.

Plant species may be transformed with the DNA construct of the presentinvention by the DNA-mediated transformation of plant cell protoplastsand subsequent regeneration of the plant from the transformedprotoplasts in accordance with procedures well known in the art. Fusionof tobacco protoplasts with DNA-containing liposomes or viaelectroporation is known in the art. (Shillito et al., “Direct GeneTransfer to Protoplasts of Dicotyledonous and Monocotyledonous Plants bya Number of Methods, Including Electroporation”, Methods in Enzymology153, pp. 3 13-36 (1987)).

As used herein, transformation refers to the introduction of exogenousDNA into cells, so as to produce transgenic cells stably transformedwith the exogenous DNA.

Transformed cells are induced to regenerate intact tobacco plantsthrough application of tobacco cell and tissue culture techniques thatare well known in the art. The method of plant regeneration is chosen soas to be compatible with the method of transformation. The stablepresence and the orientation of the QPRTase sequence in transgenictobacco plants can be verified by Mendelian inheritance of the QPRTasesequence, as revealed by standard methods of DNA analysis applied toprogeny resulting from controlled crosses. After regeneration oftransgenic tobacco plants from transformed cells, the introduced DNAsequence is readily transferred to other tobacco varieties throughconventional plant breeding practices and without undue experimentation.

For example, to analyze the segregation of the transgene, regeneratedtransformed plants (R₀) may be grown to maturity, tested for nicotinelevels, and selfed to produce R₁ plants. A percentage of R₁ plantscarrying the transgene are homozygous for the transgene. To identifyhomozygous R₁ plants, transgenic R₁ plants are grown to maturity andselfed. Homozygous R₁, plants will produce R₂ progeny where each progenyplant carries the transgene; progeny of heterozygous R₁, plants willsegregate 3:1.

As nicotine serves as a natural pesticide which helps protect tobaccoplants from damage by pests. It may therefore be desirable toadditionally transform low or no nicotine plants produced by the presentmethods with a transgene (such as Bacillus thuringiensis) that willconfer additional insect protection.

A preferred plant for use in the present methods are species ofNicotiana, or tobacco, including N tabacum, N rustica and N glutinosa.Any strain or variety of tobacco may be used. Preferred are strains thatare already low in nicotine content, such as Nic1/Nic2 double mutants.

Any plant tissue capable of subsequent clonal propagation, whether byorganogenesis or embryogenesis, may be transformed with a vector of thepresent invention. The term “organogenesis,” as used herein, means aprocess by which shoots and roots are developed sequentially frommeristematic centers; the term “embryogenesis,” as used herein, means aprocess by which shoots and roots develop together in a concertedfashion (not sequentially), whether from somatic cells or gametes. Theparticular tissue chosen will vary depending on the clonal propagationsystems available for, and best suited to, the particular species beingtransformed. Exemplary tissue targets include leaf disks, pollen,embryos, cotyledons, hypocotyls, callus tissue, existing meristematictissue (e.g., apical meristems, axillary buds, and root meristems), andinduced meristem tissue (e.g., cotyledon meristem and hypocotylmeristem).

Plants of the present invention may take a variety of forms. The plantsmay be chimeras of transformed cells and non-transformed cells; theplants may be clonal transformants (e.g., all cells transformed tocontain the transcription cassette); the plants may comprise grafts oftransformed and untransformed tissues (e.g., a transformed root stockgrafted to an untransformed scion in citrus species). The transformedplants may be propagated by a variety of means, such as by clonalpropagation or classical breeding techniques. For example, firstgeneration (or T1) transformed plants may be selfed to give homozygoussecond generation (or T2) transformed plants, and the T2 plants furtherpropagated through classical breeding techniques. A dominant selectablemarker (such as npt11) can be associated with the transcription cassetteto assist in breeding.

In view of the foregoing, it will be apparent that plants which may beemployed in practicing the present invention include those of the genusNicotiana.

Those familiar with the recombinant DNA methods described above willrecognize that one can employ a full-length QPRTase cDNA molecule or afull-length QPRTase chromosomal gene, joined in the sense orientation,with appropriate operably linked regulatory sequences, to constructtransgenic tobacco cells and plants. (Those of skill in the art willalso recognize that appropriate regulatory sequences for expression ofgenes in the sense orientation include any one of the known eukaryotictranslation start sequences, in addition to the promoter andpolyadenylation/transcription termination sequences described above).Such transformed tobacco plants are characterized by increased levels ofQPRTase, and thus by higher nicotine content than untransformed controltobacco plants.

It should be understood, therefore, that use of QPRTase DNA sequences todecrease or to increase levels of QPRT enzyme, and thereby to decreaseor increase the nicotine content in tobacco plants, falls within thescope of the present invention.

As used herein, a crop comprises a plurality of plants of the presentinvention, and of the same genus, planted together in an agriculturalfield. By “agricultural field” is meant a common plot of soil or agreenhouse. Thus, the present invention provides a method of producing acrop of plants having altered QPTRase activity and thus having increasedor decreased nicotine levels, compared to a similar crop ofnon-transformed plants of the same species and variety.

The examples which follow are set forth to illustrate the presentinvention, and are not to be construed as limiting thereof.

EXAMPLE 1 Isolation and Sequencing

TobRD2 cDNA (Conkling et al., Plant Phys. 93, 1203 (1990)) was sequencedand is provided herein as SEQ ID NO: 1, and the deduced amino acidsequence as SEQ ID NO:2. The deduced amino acid sequence was predictedto be a cytosolic protein. Although plant QPTase genes have not beenreported, comparisons of the NtPT1 amino acid sequence with the GenBankdatabase (FIG. 3) revealed limited sequence similarity to certainbacterial and other proteins; quinolate phosphoribosyl transferase(QPRTase) activity has been demonstrated for the S. typhimurium, E.coli. and N tabacum genes. The NtQPT1 encoded QPTase has similarity tothe deduced peptide fragment encoded by an Arabidopsis EST (expressionsequence tag) sequence (Genbank Accession number F20096), which mayrepresent part of an Arabidopsis QPTase gene.

EXAMPLE 2 In-Situ Hybridizations

To determine the spatial distribution of TobRD2 mRNA transcripts in thevarious tissues of the root, in situ hybridizations were performed inuntransformed plants. In-situ hybridizations of antisense strand ofTobRD2 to the TobRiD2 mRNA in root tissue was done using techniques asdescribed in Meyerowitz, Plant MoL Bid. Rep. 5,242 (1987) and Smith etal., Plant Mol. Biol. Rep. 5, 237 (1987). Seven day old tobacco(Nicotiana tabacum) seedling roots were fixed in phosphate-bufferedglutaraldehyde, embedded in Paraplast Plus (Monoject Inc., St. Louis,Mo.) and sectioned at 8 mm thickness to obtain transverse as well aslongitudinal sections. Antisense TobRD2 transcripts, synthesized invitro in the presence of 355-ATP, were used as probes. The labeled RNAwas hydrolyzed by alkaline treatment to yield 100 to 200 base massaverage length prior to use.

Hybridizations were done in 50% formamide for 16 hours at 42° C., withapproximately 5×10⁶ counts-per-minute (cpm) labeled RNA per milliliterof hybridization solution. After exposure, the slides were developed andvisualized under bright and dark field microscopy. The hybridizationsignal was localized to the cortical layer of cells in the roots(results not shown). Comparison of both bright and dark field images ofthe same sections localized TobRD2 transcripts to the parenchymatouscells of the root cortex. No hybridization signal was visible in theepidermis or the stele.

EXAMPLE 3 TobRD2 mRNA Levels in Nic1 and Nic2 Tobacco Mutants andCorrelation to Nicotine Levels

TobRD2 steady-state mRNA levels were examined in Nic1 and Nic2 mutanttobacco plants. Nic1 and Nic2 are known to regulate quinolatephosphoribosyl transferase activity and putrescence methyl-transferaseactivity, and are co-dominant regulators of nicotine production. Thepresent results are illustrated in FIGS. 5A and 5B show that TobRD2expression is regulated by Nic1 and Nic2.

RNA was isolated from the roots of wild-type Burley 21 tobacco plants(Nic1/Nic1 Nic2/Nic2); roots of Nic1-Burley 21 (nic1/nic1 Nic2/Nic2);roots of Nic2-Burley 21 (Nic1/Nic] nic2/nic2); and roots ofNic1Nic2-Burley 21 (nic1/nic1 nic2/nic2).

Four Burley 21 tobacco lines (nic) were grown from seed in soil for amonth and transferred to hydroponic chambers in aerated nutrientsolution in a greenhouse for one month. These lines were isogenic,except for the two low-nicotine loci, and had genotypes of Nic1/Nic1Nic2/Nic2, Nic1/Nic1 nic2/nic2, nic1/nic1 Nic2/Nic2, nic1/nic1nic2/nic2. Roots were harvested from about 20 plants for each genotypeand pooled for RNA isolation. Total RNA (1 [μg) from each genotype waselectrophoresed through a 1% agarose gel containing 1.1 M formaldehydeand transferred to a nylon membrane according to Sambrook et al. (1989).The membranes were hybridized with ³²P-labeled TobRD2 cDNA fragments.Relative intensity of TobRD2 transcripts were measured by densitometry.FIG. 5 (solid bars) illustrates the relative transcript levels (comparedto Nic1/Nic1 Nic2/Nic2) for each of the four genotypes. The relativenicotine content (compared to Nic1/Nic1 Nic2/Nic2) of the four genotypesis shown by the hatched bars.

FIG. 5 graphically compares the relative steady state TobRD2 5 mRNAlevel, using the level found in wild-type Burley 21 (Nic1/Nic1Nic2/Nic2) as the reference amount. TobRD2 mRNA levels in Nic1/Nic2double mutants were approximately 25% that of wild-type tobacco. FIG. 5Bfurther compares the relative levels of nicotine in the near isogeniclines of tobacco studied in this example (solid bars indicate TobRD2transcript levels; hatched bars indicate nicotine level). There was aclose correlation between nicotine levels and TobRD2 transcript levels.

EXAMPLE 4 The Effect of Topping on TobRD2 mRNA Levels

It is well known in the art that removal of the flower head of a tobaccoplant (topping) increases root growth and increases nicotine content ofthe leaves of that plant. Topping of the plant and is a standardpractice in commercial tobacco cultivation, and the optimal time fortopping a given tobacco plant under a known set of growing conditionscan readily be determined by one of ordinary skill in the art.

Tobacco plants (N tabacum SRI) were grown from seed in soil for a monthand transferred to pots containing sand. Plants were grown in agreenhouse for another two months until they started setting flowers.Flower heads and two nodes were then removed from four plants (topping).A portion of the roots was harvested from each plant after the indicatedtime and pooled for RNA extraction. Control plants were not decapitated.Total RNA (1 μg) from each time point was electrophoresed through a 1%agarose gel containing 1.1M formaldehyde and transferred to a nylonmembrane according to Sambrook, et al. (1989). The membranes werehybridized with ³²P-labeled TobRD2 cDNA fragments. Relative intensity ofTobRD2 transcripts were measured by densitometry. FIG. 6 illustrates therelative transcript levels (compared to zero time) for each time-pointwith topping (solid bars) or without topping (hatched bars).

Relative TobRD2 levels were determined in root tissue over 24 hours;results are shown in FIG. 6 (solid bars indicate TobRD2 transcriptlevels in topped plants; hatched bars indicate the TobRD2 transcriptlevels in non-topped controls). Within six hours of topping of tobaccoplants, mRNA levels of TobRD2 increased approximately eight-fold in thetopped plants; no increase was seen in control plants over the same timeperiod.

EXAMPLE5 Complementation of Bacterial Mutant Lacking OPRTase with DNA ofSEQ ID NO:1

Escherichia coli strain TH265 is a mutant lacking quinolatephosphoribosyl transferase (nadC−), and therefore cannot grow on medialacking nicotinic acids.

TH265 cells were transformed with an expression vector (pWS161)containing DNA of SEQ ID NO:1, or transformed with the expression vector(pKK233) only. Growth of the transformed bacteria was compared to growthof TH265 (pKK233) transformants, and to growth of the untransformedTH265 nadC− mutant. Growth was compared on ME minimal media (lackingnicotinic acid) and on ME minimal media with added nicotinic acid.

The E. coli strain with the QPTase mutation (nadC), TH265, was kindlyprovided by Dr. K.T. Hughes (Hughes et al., J. Bact. 175:479 (1993). Thecells were maintained on LB media and competent cells prepared asdescribed in Sambrook et al (1989). An expression plasmid wasconstructed in pKK2233 (Brosius, 1984) with the TobRD2 cDNA cloned underthe control of the Tac promoter. The resulting plasmid, pWS161, wastransformed into TH265 cells. The transformed cells were then plated onminimal media (Vogel and Bonner, 1956) agar plates with or withoutnicotinic acid (0.0002%) as supplement. TH265 cells alone and TH265transformed with pKK2233 were plated on similar plates for use ascontrols.

Results are shown in FIG. 4. Only the TH265 transformed with DNA of SEQID NO:1 grew in media lacking nicotinic acid. These results show thatexpression of DNA of SEQ ID NO:1 in TH265 bacterial cells conferred theNadC+ phenotype on these cells, confirming that this sequence encodesQPRTase. The TobRID2 nomenclature was thus changed to NtQPT1.

EXAMPLE 6 Transformation of Tobacco Plants

DNA of SEQ ID NO: 1, in antisense orientation, is operably linked to aplant promoter (CaMV 35S or TobRD2 root-cortex specific promoter) toproduce two different DNA cassettes: CaMV35S promoter/antisense SEQ IDNO: 1 and TobRD2 promoter/antisense SEQ ID NO: 1.

A wild-type tobacco line and a low-nicotine tobacco line are selectedfor transformation, e.g., wild-type Burley 21 tobacco (Nic1+/Nic2+) andhomozygous nic1−/nic2− Burley 21. A plurality of tobacco plant cellsfrom each line are transformed using each of the DNA cassettes.Transformation is conducted using an Agrobacterium vector, e.g., anAgrobacterium-binary vector carrying Ti-border sequences and the nptIIgene (conferring resistance to kanamycin and under the control of thenos promoter (nptII)).

Transformed cells are selected and regenerated into transgenic tobaccoplants (R₀). The R₀ plants are grown to maturity and tested for levelsof nicotine; a subset of the transformed tobacco plants exhibitsignificantly lower levels of nicotine compared to non-transformedcontrol plants.

R₀ plants are then selfed and the segregation of the transgene isanalyzed in R₁ progeny. RI₁ progeny are grown to maturity and selfed;segregation of the transgene among RI₂ progeny indicate which RI₁,plants are homozygous for the transgene.

1-93. (canceled)
 94. A tobacco product produced from a transgenictobacco plant of the genus Nicotiana having increased quinolatephosphoribosyl transferase (QPRTase) production relative to anon-transformed control plant, said transgenic plant comprisingtransgenic plant cells containing: an exogenous nucleic acid constructcomprising, in the 5′ to 3′ direction, a promoter operable in said plantcell a heterologous nucleic acid operably associated with said promoter;said plant exhibiting increased QPRTase production compared to anon-transformed control plant and; said heterologous nucleic acidcomprising a nucleotide sequence selected from the group consisting of:(a) the nucleotide sequence of SEQ ID NO:1; (b) a nucleotide sequencethat encodes an enzyme having the amino acid sequence of SEQ ID NO:2;(c) a nucleotide sequence having at least 80% identity with thenucleotide sequence of (a) or (b) above and that encodes a quinolatephosphoribosyl transferase; (d) a nucleotide sequence that hybridizes tothe nucleotide sequence of (a) or (b) above under stringent conditionsand that encodes a quinolate phosphoribosyl transferase enzyme; and (e)a nucleotide sequence that differs from the DNA of (a), (b), (c) or (d)above due to the degeneracy of the genetic code and that encodes aquinolate phosphoribosyl transferase enzyme.
 95. A tobacco productproduced from a transgenic tobacco plant of the genus Nicotiana havingan increased level of nicotine in leaves of said plant, wherein saidplant comprises transgenic plant cells comprises: an exogenous nucleicacid construct comprising, in the 5′ to 3′ direction, a promoteroperable in said plant cell a heterologous nucleic acid operablyassociated with said promoter; said plant exhibiting increased nicotineproduction compared to a non-transformed control plant and; saidheterologous nucleic acid comprising a nucleotide sequence selected fromthe group consisting of: (a) the nucleotide sequence of SEQ ID NO:1; (b)a nucleotide sequence that encodes an enzyme having the amino acidsequence of SEQ ID NO:2; (c) a nucleotide sequence having at least 80%identity with the nucleotide sequence of (a) or (b) above and thatencodes a quinolate phosphoribosyl transferase; (d) a nucleotidesequence that hybridizes to the nucleotide sequence of (a) or (b) aboveunder stringent conditions and that encodes a quinolate phosphoribosyltransferase enzyme; and (e) a nucleotide sequence that differs from theDNA of (a), (b), (c) or (d) above due to the degeneracy of the geneticcode and that encodes a quinolate phosphoribosyl transferase enzyme. 96.A tobacco product comprising a tobacco cell comprising an exogenousnucleotide sequence encoding a quinolate phosphoribosyl transferaseenzyme, wherein the exogenous nucleotide sequence is selected from thegroup consisting of: a) the nucleotide sequence of SEQ ID NO:1; b) anucleotide sequence that encodes the amino acid sequence of SEQ ID NO:2;c) a nucleotide sequence that hybridizes to the nucleotide sequence of(a) or (b) above under stringent conditions and that encodes quinolatephosphoribosyl transferase; d) a nucleotide sequence having at least 80%identity with the nucleotide sequence of (a) or (b) above and thatencoded quinolate phosphoribosyl transferase; and e) a nucleotidesequence that differs from the nucleotide sequence of (a), (b), (c) or(d) above due to the degeneracy of the genetic code, and that encodesquinolate phosphoribosyl transferase.
 97. A tobacco product producedfrom a transgenic plant of the genus Nicotiana having increasedquinolate phosphoribosyl transferase (QPRTase) production relative to anon-transformed control plant, wherein said transgenic plant is aprogeny of the plant of claim
 1. 98. A tobacco product produced from atransgenic plant of the genus Nicotiana having increased nicotine inleaves of said plant, wherein said transgenic plant is a progeny of theplant of claim
 2. 99. The tobacco product of claim 1, wherein thetobacco product is selected from the group consisting of a cigarette,cigarette tobacco, cigar tobacco, a cigar, pipe tobacco, chewingtobacco, leaf tobacco, shredded tobacco and cut tobacco.
 100. Thetobacco product of claim 2, wherein the tobacco product is selected fromthe group consisting of a cigarette, cigarette tobacco, cigar tobacco, acigar, pipe tobacco, chewing tobacco, leaf tobacco, shredded tobacco andcut tobacco.
 101. The tobacco product of claim 3, wherein the tobaccoproduct is selected from the group consisting of a cigarette, cigarettetobacco, cigar tobacco, a cigar, pipe tobacco, chewing tobacco, leaftobacco, shredded tobacco and cut tobacco.
 102. The tobacco product ofclaim 4, wherein the tobacco product is selected from the groupconsisting of a cigarette, cigarette tobacco, cigar tobacco, a cigar,pipe tobacco, chewing tobacco, leaf tobacco, shredded tobacco and cuttobacco.
 103. The tobacco product of claim 5, wherein the tobaccoproduct is selected from the group consisting of a cigarette, cigarettetobacco, cigar tobacco, a cigar, pipe tobacco, chewing tobacco, leaftobacco, shredded tobacco and cut tobacco.